1. Field of the Invention
This invention relates to a signal processing method in autoradiography and more particularly, to a signal processing method for comparing and identifying the resolved positions of radioactively labeled substances in autoradiography for determination of base sequences of DNA and DNA fragment employing a radiosensitive material.
2. Description of the Prior Art
Autoradiography has been known as a method for obtaining locational information on radioactively labeled substances distributed in at least a one dimensional direction to form a row on a support medium.
For instance, autoradiography comprises steps of: labeling organism-originating biopolymers such as proteins or nucleic acids with a radioactive element; resolving the radioactively labeled biopolymers, derivatives thereof, or cleavage products thereof (hereinafter referred to as "radioactively labeled substances") on a gel support (support medium) through a resolving process such as electrophoresis to form a resolved pattern of the radioactively labeled substances (the resolved pattern is not visible); placing said gel support and a high-sensitivity type X-ray film together in layers for a certain period of time to expose the film and developing said film to obtain the autoradiograph of the resolved pattern as a visible image on the film; and obtaining the locational information of the radioactively labeled substances from said visible image. Further, the identification of the polymeric substances, determination of molecular weight of the polymeric substances and isolation of the polymeric substances can be performed based on the obtained locational information.
The autoradiography has been effectively utilized for determining the base sequence of nucleic acids such as DNA (or DNA fragments, hereinafter "DNA" is used to include DNA as well as DNA fragments) or the like.
The Maxam-Gilbert method and Sanger-Coulson method are known as methods for sequencing DNA utilizing autoradiography. In these methods, the base sequence of DNA is determined by utilizing the characteristic structure of DNA in that DNA is in the form of double helix structure consisting of two chain molecules, which are constituted by four constitutional base units, each unit having a base, namely adenine (A), guanine (G), thymine (T) or cytosine (C), and cros-linked by hydrogen bonding between the four bases, the hydrogen bonding between each constitutional base unit comprising only two combinations, namely G-C and A-T.
For instance, the Maxam-Gilbert method is carried out as follows: a group containing a radioactive isotype of phosphorus (P) is attached to a chain molecule of DNA or a DNA fragment at one end to be sequenced to prepare a radioactively labeled DNA molecule, and then the thus labeled DNA molecule is specifically cleaved at the constitutional base units by a certain chemical reactions. This reaction is called a "base specific cleavage reaction". Then the obtained mixture of numerous cleavage products of the DNA or DNA fragment is resolved through gel electrophoresis to give a resolved pattern of the numerous cleavage products (the pattern is not visible). An X-ray film is exposed to the resolved pattern and developed to obtain a visualized autoradiograph thereon, and the sequential position of each base from the radioisotopically labeled end of the chain molecules is read by referring to both the obtained autoradiograph and the applied base-specific chemical reaction so as to determine the sequence of all bases in the substance.
In the autoradiography utilizing the radiographic process, the visualization of the autoradiograph having locational information on radioactively labeled substances on a radiographic film is essentially required.
Investigators analyze the distribution of the radioactively labeled substances on a support medium through observation of the visualized autoradiograph. The sequence of DNA is determined by studying individual resolved positions of the radioactivity labeled cleavage products (or mixture of cleavage products) of DNA on the visualized autoradiograph, and then comparing the resolved positions among the resolved rows thereof.
Since the autoradiography requires the visual analysis of the autoradiograph, there is a drawback in that the locational information on the radioactively labeled substances obtained by analysis of the visualized autoradiograph varies or fluctuates depending on the skill of investigators, and the accuracy of the information is limited to a certain extent. Particularly, in such cases that the autoradiograph visualized on a radiographic film shows an image of reduced quality (in regard of sharpness, contrast, etc.), the satisfactory information can be hardly obtained and the accuracy is low. In order to improve the accuracy of the locational information, for instance, a visualized autoradiograph can be scanned with a device such as a scanning densitometer. However, such scanning process requires increased operation time and complicated procedures. Further, there is a limitation on increase of the accuracy when using this device.
For instance, in carrying out the exposing procedure, the support medium carrying the above-mentioned resolved rows thereon an a radiographic film sometimes cannot be accurately arranged together in layers. In such a case, the resolved rows (e.g., electrophoretic rows) visualized on the radiographic film are rendered not parallel to the longitudinal direction of the film to give a dislocated pattern. As a result, error is introduced into the visual analysis of the locational information on the radioactively labeled substances to decrease the accuracy thereof.
Further, the rows of the resolved radioactively labeled rows on the support medium are sometimes made non-parallel to the longitudinal direction of the support medium or made distorted, depending on the kind of the support medium or resolving conditions. For instance, a gel support medium is generally held between two glass plates in the resolving procedure because the gel lacks self-supporting property. As a result, the gel occasionally becomes uneven in thickness due to the deformation of the covers such as the glass plates and accordingly the radioactively labeled substances are not always resolved uniformly on the gel. The lack of uniformity of the resolved pattern is also caused by air foams contained in the gel or by heterogenous dispersion of the composition of the gel. For these reasons, a phenomenon such as the so-called smiling effect, i.e., a phenomenon that the migration distance of the resolved row in the vicinity of the center of the support medium is longer than those in both sides thereof, is often observed. Additionally, if the voltage is not applied uniformly to the support medium in the electrophoresis, the resolving conditions are made locally uneven on the support medium to distort the resolved rows.
There is known no suitable method but a method of manually correcting the distortion of resolved rows. Accordingly, it is not easy to analyze the locational information on the radioactively labeled substances in the above-described cases. Even if the aforementioned device is used, it is still difficult to obtain satisfactorily accurate locational information on the radioactively labeled substances.